The developmental sequence of cell type specific patterns of creatine phosphokinase (CPK) isoenzymes will be examined in homogeneous populations of myoblasts and fibroblasts cultured under specific conditions which promote either rapid proliferation or the maintenance of cells in a protracted Gl phase. Previously established culture protocols which reproduce the accumulation of CPK specific activity and the isoenzyme pattern characteristic of mature muscle cells will be employed to study the rates of synthesis of the individual muscle (M) and brain-type (B) CPK subunits. Cells will be pulsed with 35S-methionine at the pre-fusion stage and at regular intervals during the period of synchronous fusion. Cell extracts will be resolved on two-dimensional gels and the individual subunits identified (using purified MM and BB-CPK as markers), and quantitated. These measurements will identify the time of initiation of synthesis of the M subunit during myogenesis and the relationship of the accumulation of enzyme activity to actual subunit synthesis. While the accumulation of the homodimeric MM isoenzyme is initiated in cells blocked in Gl (viz. multinucleated muscle fibers) or in cells in a protracted Gl, (fusion-blocked skeletal myocytes), the proliferating myoblast contains only the BB isozyme. The relationship of the specific CPK subunit expressed to location in the cell cycle will be examined by first mapping enzyme activity using a microfluorimetric assay. The rate of accumulation of CPK molecules will then be examined during those cell cycle phases in which transient increases in activity occur. This data should provide a basis for judging whether cell-type specific protein patterns reflect some program of gene activity intrinsic to the cell cycle and differentially expressed dependent on the point in Gl or Go in which the specific cell eventually blocks.